Head and neck squamous cell carcinoma (HNC) is a disease that affects over 50,000 patients every year. A hallmark of aggressive HNC is its propensity to spread along nerves, a process termed perineural invasion (PNI) that is found in up to 40% of patient. It has been demonstrated that patients with PNI have significantly higher rates of locoregional recurrence and lower overall survival. Although HNC patients with PNI often receive all of the currently available therapy (surgery, chemotherapy, and radiotherapy), the overall survival rates of these patients are still significantly lower than those without PNI. Attempts at developing novel agents that target PNI have been hampered by the lack of understanding of the factors that regulate this process. Such efforts have been further hindered by the lack of in vitro models that can reliably recapitulate the major features of PNI by HNC cells. In order to bride this gap, we have recently developed a novel in vitro model of PNI based on murine dorsal root ganglia. To our knowledge, this neurite-tumor cell interaction (NTI) assay is the only assay that has successfully reproduced the invasion and migration of HNC cells along cultured neurites. Utilizing the NTI assay, we have identified neuregulin 1(NRG1), a known ligand of HER3 (a member of the ErbB receptor tyrosine kinase family that also include epidermal growth factor receptor (EGFR) and HER2), as a mediator of the HNC cell-neurite interaction. Our findings showed that HNC cells express HER3 and that the activation of HER3 by NRG1 results in the formation of HER2:HER3 heterodimers. This is followed by the activation of Src and focal adhesion kinase (Fak), and by the increased migration and invasion of the HNC cells. Co-culture of HNC cells and neurons also resulted in increased migration and invasion of HNC cell that was dependent on HER3. More importantly, the NTI assay showed that the attraction and movement of HNC cells to and along neurites is inhibited by HER3 antibodies. Therefore, we hypothesize that PNI in HNC is mediated by the paracrine activation of HER3 on tumor cells by neuronal NRG1 where HER3 targeting may be an effective therapeutic strategy. We will test this hypothesis with the following two separate but interrelated aims: i) to determine the importance of NRG1:HER3 axis in mediating PNI in HNC, and ii) to determine the whether HER2 blockade is essential for HER3 targeted inhibition of PNI. In order to study these aims, we will take advantage of our unique NTI assay, availability of tumor specimens from our institutional HNC tumor bank, and clinical available HER2 and HER3 antibodies. By demonstrating NRG1 and HER3 to be a mediator of PNI in HNC, we hope to elucidate a therapeutic target for a clinical problem against which we have no effective therapy. The completion of this study will also lay the foundation for future clinical trials of anti-ErbB agent in improving the prognosis of HNC patients with PNI.